Alternative method to visualize receptor dynamics in cell membranes

There is a close relation between membrane receptor dynamics and their behavior. Several microscopy techniques have been developed to study protein dynamics in live cells such as the Fluorescence Recovery After Photobleaching (FRAP) or the Single Particle Tracking (SPT). These methodologies require expensive instruments, are time consuming, allow the analysis of small portion of the cell or an extremely small number of receptors at a time. Here we propose a time-saving approach that allows to visualize the entire receptor pool and its localization in time. This protocol requires an epifluorescence microscope equipped for structured illuminated sectioning and for live cell imaging. It can be applied to characterize membrane receptor and multi protein complex and their response to activators or inhibitors. Image acquisition and analysis can be performed in two days, while cells and substratum preparation require a few minutes a day for three days.


•
The day before transfection, seed 2x104/cm2 of CHO cells in 6 well plate in F12 medium supplemented with 100 IU/mL penicillin and 100 µg/mL streptomycin and 10% FCS (complete medium) and culture under normal conditions at 37 °C in 5% CO2.When using different cell lines, ensure a cell density to allow 60-70% of confluence the day of the transfection.

•
After 24 hours, transfect each well of CHO cells with 4 µg of pBE-hVEGFR2-eYFP and 8 ng of PEI (1µg/µL) in serum and antibiotics free F12 medium.After 4 hours, replace medium with complete medium.

•
Incubate the 2 well chambered glass coverslips with 100 μL of sterile PBS containing 2 μg/mL of human VEGF-A for 16 hours at 4°C.The coating is carried out by placing a drop containing the recombinant protein in the center of the well in order to obtain a 10 mm diameter coating spot.VEGF-A can be replaced by other ligands able to recruit other specific receptors.

•
After 16 hours, remove unbound ligand and wash the coverslips 3 times with cold and sterile PBS.
• Block nonspecific binding sites with 1 mg/mL of BSA for 1 hour at room temperature.Under these conditions, ligand binds to the coverslip in a dose-dependent manner, with maximal binding at coating concentrations ≥ 2 μg/mL.Using this concentration, it is possible to have a spot with a high concentration of ligands.Substratum-immobilized ligand is resistant to high molar salt (2 mol/L NaCl) and detergent (0.2% Triton X-100) washes [1,2.3 Cell preparation (day 3)

•
Put glass coverslips on the bottom of a 24 well plate and ensure it remains to the bottom of the well while seeding the cells.
• 24 hours after cell transfection, plate CHO at the density of 75.000/cm2 in complete medium on the coverslips and culture under normal conditions for 16 hours.When using different cell lines, ensure a cell density to allow 50-80% of confluence the day of image acquisition.4 Image acquisition (day 4)

•
Replace the complete medium of transfected cells with F12 (without phenol red) 1% FCS and culture under normal conditions for 2 hours.• After 2 hours of starvation, flip upside-down the cell-plated coverslips on immobilized-VEGF chambered in F12 1% FCS.

•
Put the sample in the microscope incubator at 37 °C and 5% CO2.
To analyze VEGFR2 recruitment we acquired Z-stack images for 120 minutes.
• Acquire images using YFP fluorescence filter set (excitation: 500/20; dichroic: long pass 512; emission: 535/30).CRITICAL STEP: It is very important to acquire all the images with the same camera exposure.This allows you to compare different images or different experimental conditions, when necessary.

•
Acquire imaging with a PlanApochromat 63X/1.4NAOil objective and Apotome structured illumination that allow a sectioning of 1.3 µm.Set an overlap of 50% between two consecutive stacks.On average, a whole CHO cell is acquired in 10-12 slices, with a total thickness of acquisition of 13-15.6 µm.

•
Process images without deconvolution.
5 Image quantification and data analysis (day 5) Open the Threshold menu and set threshold manually in order to clearly see the specific fluorescence standing out from the background.CRITICAL STEP: It is very important to set the same threshold for all the images.This allows you to compare different images or different experimental conditions, when necessary.• Create a 3D image using "3D viewer " plugin.

•
Draw, using freehand selection, the projection of the cell.Analyze one cell at a time.Scroll through the image sequence measuring Area and Area percentage in every stack.• Save data for the analysis.• Calculate the number of pixels positive for VEGFR2 associated fluorescence using the formula: N° of VEGFR2-positive pixels = Area percentage * (Area/100) • Sum all the pixel from each Z-stack to obtain the total amount of VEGFR2-positive pixels for cell • Calculate the distribution of VEGFR2 in each stack using the formula: % of VEGFR2 area = ( N° of VEGFR2-positive pixels / total amount of VEGFR2-positive pixels) * 100 Note that the sum of % of VEGFR2 area from all the stacks should be 100.6 3D reconstruction • Select a region of interest (ROI) that includes one cell or more.Save the image.• Create orthogonal projection by choosing, from Image > Stacks the Orthogonal Views command.